The invention relates to the identification of a novel Varicella-Zoster Virus (hereinafter to be referred to as VZV) gene. It also relates to methods of making and to novel immunogenic compositions comprising VZV, modified by the modification or deletion of this novel gene and to methods inducing an immune response in a host organism by administration of such immunogenic compositions.
Varicella-Zoster Virus, a member of the herpes virus family, causes chicken pox as a primary infection and shingles as a secondary infection. Chicken pox is a common disease of children that is highly contagious but usually not life threatening. In adults chicken pox can pose a more serious medical threat. Shingles occurs after primary infection with the vaccine or the wild type virus. After the primary infection, the virus travels to the trigeminal or thoracic (commonly T3-L2) ganglia and lies dormant. Upon reactivation, the virus affects the dermatome supplied by the corresponding ganglia. Both of these diseases are widespread, with 3-4 million varicella and 850,000 zoster cases in the U.S. per year, and costs $700 million per year in the U.S. in treatment and lost work costs.
Chemotherapy is available to combat VZV infections. Acyclovir chemotherapy halts the progression of disease by acting as a substrate for herpes pyrimidine deoxyribonucleotide kinase. The kinase phosphorylates Acyclovir, and the phosphorylated Acyclovir is then incorporated into viral DNA, which ultimately leads to DNA chain termination. Although chemotherapy is effective in treating infected patients with varicella or zoster symptoms it does not prevent occurrence of either disease.
An alternative post-infection treatment, VZV-specific immunoglobulin, is available, in addition to chemotherapy, to treat VZV infections. However, this treatment is expensive, affords only a narrow window of therapeutic use and must be quality controlled for HIV contamination.
A live, attenuated vaccine made of VZV derived from the Oka strain of that organism has been developed by Takahashi, M., et al., Lancet 1974; 2:1288-90. The vaccine was developed by multiple passage of the virus through a mammalian cell line. It protects against chicken pox in humans. However, zoster infections resulting from Oka replication have been documented in humans vaccinated with the Oka vaccine. Unfortunately, no other vaccines against VZV are currently marketed.
Progress toward developing a VZV vaccine has been stymied because no practical animal model exists for determining vaccination and immune responses and because only low viral yields in culture are currently available. Guinea pigs, and perhaps marmosets, can be used as limited models for VZV studies. Infection can be documented in these animals, although latency and reactivation of the virus has yet to be established. One 6 month old gorilla has been shown to be infected with a VZV indistinguishable from a human strain. Even though the disease closely resembled that of human varicella, the gorilla is an impractical disease model for obvious socio-economic reasons. Although culture yields have been improved somewhat by growing attenuated vaccines in monolayers (see for example PCT Patent Application WO93/24616 published Dec. 9, 1993), higher yields would be even more advantageous. Additionally, deletions in the VZV genome to produce attenuated strains of VZV which would make good candidates for live vaccines have yielded varying results. For example, deletion of the gene encoding ribonucleotide reductase slowed viral growth and increased Acyclovir sensitivity. In contrast, prevention of VZV thymidylate synthetase gene expression failed to affect viral growth rates.
As noted previously, VZV is a member of the herpesvirus family. As such, the 120 kilobase (kb) VZV genome shares some structural homology to herpes simplex virus. Both viruses store their virion DNA as a linear molecule and replicate using circular DNA molecules. The linear order of genes within the each DNA molecule is similar for most genes in both herpes simplex virus and VZV. Each genome contains two unique sequences. UL (standing for xe2x80x9cunique longxe2x80x9d) comprises about 100 kb and US (standing for xe2x80x9cunique shortxe2x80x9d) comprises about 5 kb. Each UL sequence is flanked by a terminal repeat sequence, i.e., a TRL (or terminal repeat of unique long), and an internal repeat sequence, i.e., an IRL (internal repeat of unique long). Likewise, each US sequence is flanked by a terminal repeat sequence, TRS, and an internal repeat sequence, IRS.
However, VZV and herpes simplex virus display numerous differences in their genomic structure as well. The herpes simplex virus genome contains an extra set of inverted repeats called the xe2x80x9caxe2x80x9d sequence, in addition to TRL, IRL, TRS and IRS sequences. The a sequences are located at both genomic termini, as well as at the junction of the L and S components. The VZV TRL and IRL sequences are very short (88 base pairs) compared to the herpes simplex virus TRL and IRL sequences (8000 base pairs). Another difference is that there is an origin of DNA replication at the approximate center of the UL sequence in herpes simplex virus. There is no such structure in VZV. The functional and evolutionary significance of the differences between the repeat regions flanking the UL region in herpes simplex virus and the repeat regions flanking the UL region in VZV is unknown.
Four different genomic isomers, composed of Par (Parental) and inverted (Inv) forms of the UL and US repeat sequences, exist in the VZV genome: UL-Par/US-Par, UL-Par/US-Inv, UL-Inv/US-Par, and UL-Inv/US-Inv. The four isomers are not randomly distributed. Two isomers (UL-Par/US-Par and UL-Par/US-Inv) account for 95 % of the packaged DNA and the remaining two isomers (ULInv/US-Par and UL-Inv/US-Inv) account for 5 % of the packaged DNA, as shown in FIG. 1.
The VZV genome contains 80 possible open reading frames (hereinafter abbreviated as ORF or ORFs), although fewer genes, approximately 70, are thought to encode gene products used in the viral replication cycle. The ORF""s of VZV are based on ORF criteria from Davison, A. J. and Scott, J. E., Journal of General Virology (1983); 64:1811-184 (ORFs with a methionine initiation site and at least 150 amino acids or ORFs with a TATA box, no overlap with other ORFs and good codon usage). Little is known about the function of the approximately twenty known VZV gene products. The novel gene identified herein was not detected by Davison and Scott type ORF criteria.
Some of the VZV gene products lacking a demonstrated function show nucleic acid or amino acid sequence homology to herpes simplex virus genes of known function. As used herein, xe2x80x9chomologxe2x80x9d or xe2x80x9chomologousxe2x80x9d refers solely to nucleic acid or protein sequence homology between two sequences from different organisms, and does not encompass any functional similarity. About 62 of the known VZV genes have homologs in the herpes simplex virus genome. Five known VZV genes have no homologs in the herpes simplex virus genome. Homologs are traditionally determined using computer sequence analysis methods or using nucleotide probing of nucleotide sequences, methods that require a level of sequence homology sufficient to allow recognition of homologs above background. The novel ORFS/L gene disclosed herein for the first time had not been previously detected using either of the traditional methods. Surprisingly, it has been found that this novel gene is actually a positional homolog of the xcex3134.5 gene of herpes simplex. It apparently previously escaped detection because it lacks a typical TATA consensus element upstream of its open reading frame, it has an unusual and unexpected gene structure and it is located in an unexpected location of the VZV genome.
The role of herpes simplex virus xcex3134.5 gene in neuro-virulence and growth was disclosed in Science; 250:1262-1266 (1990), xe2x80x9cMapping of Herpes Simplex Virus-1 Nuro-virulence to xcex3134.5, a gene Nonessential for Growth in Culturexe2x80x9d by J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman.
The existence of a herpes simplex virus gene product near the internal repeat region of herpes simplex virus genome was disclosed in the Journal of Virology. 57:629-637 (1986), xe2x80x9cThe Terminal a Sequence of the Herpes Simplex Virus genome contains the Promoter of a Gene Located in the Repeat Sequences of the L Componentxe2x80x9d by J. Chou and B. Roizman.
It has been disclosed that herpes simplex viral mutants, lacking a functional xcex3134.5 gene, expressed early proteins, but viral DNA synthesis resulted in the cessation of viral protein synthesis. Proc. Natl. Acad. of Sci. USA. 89:3266-3270 (1992), xe2x80x9cThe xcex3134.5 gene of herpes simplex virus 1 precludes . . . xe2x80x9d by J. Chou and B. Roizman.
It has been disclosed that the Oka strain of VZV can be reconstructed using overlapping cosmid clones and that mutants, lacking a functional thymidylate synthetase gene, grew at a rate similar to the wild type virus. Pro. Natl. Acad. of Sci. USA. 90:7376-7380 (1993), xe2x80x9cGeneration of Varicefla-Zoster Virus (VZV) and viral mutants from cosmid DNAs: . . . xe2x80x9d by J. L. Cohen and K. E. Seidel.
The introduction of a stop codon into the thymidylate synthetase gene resulted in the lack of thymidylate synthetase protein expression without affecting viral growth rates or Acyclovir sensitivity, as disclosed in XVII International Herpesvirus Workshop Abstract 1993, xe2x80x9cGeneration of Varicella-Zoster Virus and Viral Mutants from Cosmic DNA; Thymidylate Synthetase is Not Essential for Replication In Vitroxe2x80x9d by Jeffrey I. Cohen and Karen E. Seidel.
For a general review of VZV see Journal of Virology 72:475-486 (1991), xe2x80x9cVaricella-Zoster Virusxe2x80x9d by A. J. Davidson.
The DNA sequence and open reading frames of VZV are disclosed in Journal of General Virology. 67:1759-1816 (1986), xe2x80x9cThe Complete DNA Sequence of VariceUa-Zoster Virusxe2x80x9d by A. J. Davison and J. E. Scott.
A bovine Herpes virus gene that is formed during the circularization of a bovine Herpes virus genome was disclosed in the Journal of Virology; 67:1328-1333 (1993), xe2x80x9cImmediate-Early Transcription over Covalently Joined Genome Ends of Bovine Herpesvirus-1: the circ Genexe2x80x9d by C. Fraefel, et al.
The deletion of VZV ORF""s for ribonucleotide reductase slowed viral growth rate, increased Acyclovir sensitivity and reduced plaque size, as disclosed in XVIII International Herpes Workshop Abstract 1993, xe2x80x9cProduction and Characterization of a VZV Mutant Lacking the Large Subunit of Ribonucleotide Reductasexe2x80x9d by T. C. Heineman and J. I. Cohen.
The immune response of elderly individuals to the Oka vaccine is discussed in The Journal of Infectious Diseases; 166:253-9 (1992), xe2x80x9cImmune Response of Elderly Individuals to a Live Attenuated Varicella Vaccinexe2x80x9d by Levin, M. J., et al.
The hairless guinea pig model is disclosed in The Journal of Infectious Diseases; 163:746-751 (1991), xe2x80x9cVaricella in Hairless Guinea Pigsxe2x80x9d by Myles, M. G., et al.
Infection of a gorilla by varicella was disclosed in Journal of Medical Virology, 23:317-322 (1987), xe2x80x9cVaricella in Gorillaxe2x80x9d by Myer, M. G. et al.
Lastly, U.S. Pat. No. 4,686,101 (1987); U.S. Pat. No. 4,769,239 (1988), U.S. Pat. No. 4,812,559 (1989) and U.S. Pat. No. 4,952,674 (1990) issued to Ellis and Keller disclose the isolation and cloning of the gC glycoprotein of VZV and their use in vaccines against VZV.